Throughout the application various publications are referenced in parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in the application in order to more fully describe the state of the art to which this invention pertains.
1. Field of the Invention
The present invention relates to a method of inhibiting neoplastic cellular proliferation and/or transformation of mammalian cells, in vitro and in vivo.
2. Related Art
Pituitary Tumor Transforming Gene (PTTG) is highly expressed in pituitary tumors and neoplasms from the hematopoietic system and colon. (Zhang, X. et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endocrinol. 13: 156-66 [1999a]; Zhang, X. et al., Pituitary tumor transforming gene (PTTG) expression in pituitary adenomas, J. Clin. Endocrinol. Metab. 84:761-67 [1999b]; Heaney, A. P. et al., Pituitary tumor transforming gene in colorectal tumors, Lancet 355:712-15[2000]; Dominguez, A. et al., hpttg, a human homologue of rat pttg, is overexpressed in hematopoietic neoplasms. Evidence for a transcriptional activation function of hPTTG, Oncogene 17:2187-93 [1998]; Saez, C. et al., hpttg is over-expressed in pituitary adenomas and other primary epithelial neoplasias, Oncogene 18:5473-6 [1999]). PTTG1 is expressed at low levels in most normal human tissues. (Chen, L. et al., Identification of the human pituitary tumor transforming gene (hPTTG) family: molecular structure, expression, and chromosomal localization, Gene. 248:41-50 [2000]; Heaney, A. P. et al. [2000]).
Levels of PTTG expression positively correlate with pituitary and colorectal tumor invasiveness (Zhang, X. et al. [1999b]; Heaney, A. P. et al. [2000]) and are induced by estrogen. (Heaney, A. P. et al., Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis, Nat. Med. 5:1317-21 [1999]). In tumor cells, PTTG mRNA and protein expressions are cell cycle-dependent and peak at G2/M phase. (Yu, R. et al., Pituitary Tumor Transforming Gene (PTTG) Regulates Placental JEG-3 Cell Division and Survival. Evidence from Live Cell Imaging, Mol. Endocrinol. 14:1137-1146 [2000]). The mechanism of PTTG action is not very clear. PTTG upregulates basic fibroblast growth factor secretion (Zhang, X. et al. [1999a]), and transactivates DNA transcription (Dominguez, A. et al. [1998]; Wang, Z. et al., Pituitary tumor transforming gene (PTTG) transactivating and transforming activity, J. Biol. Chem. 275:7459-61[2000]).
PTTG encodes a securin protein the expression of which causes cell transformation, induces the production of basic fibroblast growth factor (bFGF), is regulated in vitro and in vivo by estrogen, and inhibits chromatid separation. (Pei, L., and Melmed S., Isolation and characterization of a pituitary tumor transforming gene, Mol. Endocrinol. 11:433-441 [1997]; Zhang, X., et al., Structure, expression, and function of human pituitary tumor-transforming gene (PTTG), Mol. Endocrinol. 13:156-166 [1999a]; Heaney, A. P., et al., Early involvement of estrogen-induced pituitary tumor transforming gene and fibroblast growth factor expression in prolactinoma pathogenesis, Nature Med. 5:1317-1321 [1999]; Zou, H., et al., Identification of a vertebrate sister-chromatid separation inhibitor involved in transformation and tumorigenesis, Science 285:418-422 [1999]).
By dysregulating chromatid separation, PTTG overexpression also leads to aneuploidy, i.e., cells having one or a few chromosomes above or below the normal chromosome number. (Zou et al. [1999]; Yu, R. et al. [2000]). At the end of metaphase, securin is degraded by an anaphase-promoting complex, releasing tonic inhibition of separin, which in turn mediates degradation of cohesins, the proteins that hold sister chromatids together. Overexpression of a nondegradable PTTG disrupts sister chromatid separation (Zou et al. [1999]) and overexpression of PTTG causes apoptosis and inhibits mitosis (Yu, R. et al. [2000]). The securin function of PTTG suggests that PTTG may also be expressed in normal proliferating cells. In adult animals and humans, PTTG mRNA is most abundant in testis (Zhang, X. et al. [1999a]); Wang, Z. et al. [2000]), an organ containing rapidly proliferating gametes.
A PTTG gene family contains at least three genes that share a high degree of sequence homology, including human PTTG1, located on chromosome 5q33. (Prezant, T. R., et al., An intronless homolog of human proto-oncogene hPTTG is expressed in pituitary tumors: evidence for hPTTG family, J. Clin. Endocrinol. Metab. 84:1149-52 [1999]). Murine PTTG shares 66% nucleotide base sequence homology with human PTTG1 and also exhibits transforming ability. (Wang, Z. and Melmed, S., Characterization of the murine pituitary tumor transforming gone (PTTG) and its promoter, Endocrinology [In Press; 2000]). A proline-rich region was identified near the protein C-terminus that is critical for PTTG1's transforming activity. (Zhang, X., et al. [1999a]), as demonstrated by the inhibitory effect on in vitro transformation, in vivo tumorigenesis, and transactivation, when point mutations were introduced into the proline-rich region. Proline-rich domains may function as SH3 binding sites to mediate signal transduction of protein-tyrosine kinase. (Pawson, T., Protein modules and signaling networks, Nature 373:573-580 [1995]; Kuriyan, J., and Cowburn, D., Modular peptide recognition domains in eukaryotic signaling, Annu, Rev. Biophys. Biomol. Struct. 26:259-288 [1997]).
Breast and ovarian cancers are a model of hormone dependent malignancy. Estrogens and progesterone, acting via specific nuclear receptors, are necessary for normal development of mammary gland and ovarian tissue and their differentiated function. In addition to classical estrogenic ligand-estrogen receptor (ER) interactions, and subsequent ER binding to estrogen-response elements to regulate gene transcription, it is now apparent that transcriptional modulation can be mediated through the membranal ER. (Levin E. R., Cellular functions of the plasma membrane estrogen receptor, TEM 10:374-77 [1999]). This action requires modification of cytosolic signal transduction pathways such as extracellular-signal-regulated kinase/mitogen-activated protein kinase pathways (ERK/MAPK).
In breast and ovarian cancers, the molecular mechanisms through which these signal transduction effects are mediated are not well defined, although c-myc and cyclin D1 have been identified as major downstream targets of estrogen and progestin-stimulated cell cycle progression. In addition to regulating cyclin abundance, recruitment of specific CDK inhibitors, such as p21 is impaired by estrogen, and additional, as yet undefined estrogen-regulated components are likely to be regulators of mammary epithelial cell proliferation and differentiation. (Sutherland, R. L., et al., Estrogen and progestin regulate cell cycle progression, J. Mammary Gland Biol. Neoplasia 3:63-72 [1998]).
Several studies have described the involvement of SP1 and half-site EREs in conferring estrogen-responsiveness of several genes, including creatine kinase B, c-myc, the retinoic acid receptor α, heat shock protein 27. (Wu-Peng X. et al., Delineation of sites mediating estrogen regulation of the rat creatine kinase B gene, Mol. Endocrinol. 6:231-240 [1992]; Dubik, D. and Shiu, R., Mechanism of estrogen activation of c-myc oncogene expression, Oncogene 7:1587-1594 [1992]). This cooperative interaction of a half-site ERE and an SP1 site has recently been described for the progesterone receptor (Petx, L. and Nardulli, A. M., Sp1 binding sites and an estrogen response element half-site are involved in regulation of the human progesterone receptor A promoter, Mol. Endocrinol. 14:972-85 [2000]). In the context of complex promoters, EREs are generally found in multiple copies or encased among binding motifs for other transcription factors (Porter, W. et al., Functional synergy between the transcription factor Sp1 and the estrogen receptor, Mol. Endo. 11:1569-80 [1997]). It has been demonstrated that the SP1 sites on the murine and human PTTG-promoter are crucial for its transactivation activity, and mutational disruption of the SP1 element or competition with a known SP1 oligo resulted in up to 90% loss of PTTG-promoter activity. (Wang, Z. and Melmed, S., SP1 activates the pituitary tumor transforming gene (PTTG) promoter during cellular transformation J Biol Chem [2000]; Kakar, S. S., Molecular cloning, genomic organization, and identification of the promoter for the human pituitary tumor transforming gene (PTTG), Gene 240: 317-324 [1999]).
In many solid tumors, tumor vascularity may inversely correlate with prognosis, and both bFGF and VEGF expression have been reported to predict prognosis (Takahashi, Y. et al., Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer, Cancer Res 55:3964-68 [1995]). Quantification of angiogenesis in breast cancer can be used as an independent prognostic factor. (Weidner, N. et al., Tumor angiogenesis: a new significant and independent prognostic factor in early-stage breast carcinoma, J. Natl. Cancer Inst. 84:1875-1887 [1992]; Horak, E. R. et al., Angiogenesis, assessed by platelet/endothelial cell adhesion molecule antibodies, as indicator of node metasteses and survival in breast cancer, Lancet 340:1120-1124 [1992]). Not only are tumor growth, progression, and metastasis dependent on access to vasculature, but it is also apparent that during the transition from mid-late dysplasia, as in the case of cervical intraepithelial neoplasia II (CIN II) to CIN III, an “angiogenic switch” is activated and changes in tissue angiogenic phenotype probably precede the histological tissue transition. (Hanahan, D. and Folkman, J., Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 86:353-64 [1996]).
The sequence of events in angiogenesis leading to formation of new blood vessels from pre-existing vessels is highly regulated (Jain, R K et al., Quantitative angiogenesis assays: progress and problems, Nat Med. 3:1203-1208 [1997]; Darland D C and D'Amore P A, Blood vessel maturation: vascular development comes of age, J Clin Invest. 103:157-158 [1999]), and involves dissolution of vessel basement membranes, and formation of new lumen and pericytes by vascular endothelial cells. During tumor-associated angiogenesis, sustained production of angiogenic factors by cancer cells, or indirect macrophage stimulation, causes disregulated immature vessel growth (Folkman, J. and Shing, Y., Angiogenesis, J Biol Chem. 267:10931-10934[1992]). A number of in vitro and in vivo assays have been useful for studying angiogenesis (e.g., Jain, R K et al. [1997]; Auerbach, R. et al., Assays for angiogenesis: a review, Pharmacol Ther. 51:1-11 [1991]).
Several cytokines and growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) modulate angiogenesis in vivo with a paracrine mode of action. (Bikfalvi, A. et al., Biological roles of fibroblast growth factor-2, Endocr. Rev. 18:26-45 [1997]; Ferrara, N. and Davis-Smyth, T., The biology of vascular endothelial growth factor, Endocr Rev 18:4-25 [1997]). bFGF and VEGF levels in cytosolic fractions are significantly associated with intratumoral vascularization. (Expression of the angiogenic factors vascular endothelial cell growth factor, acidic and basic fibroblast growth factor, tumor growth factor-1, platlet-derived endothelial cell growth factor, placenta growth factor, and pleiotrophin in human primary breast cancer and its relation to angiogenesis, Cancer Res. 57:963-69 [1997]; Linderholm, B. et al., Vascular endothelial growth factor is of high prognostic value in node-negative breast carcinoma, J. Clin. Oncol. 16:3121-28 [1998]). bFGF and VEGF have synergistic effects on angiogenesis, and bFGF modulates endothelial expression of VEGF through both autocrine and paracrine actions (Seghezzi, G. et al., Fibroblast growth factor-2 (FGF-2) induces vascular endothelial growth factor (VEGF) expression in the endothelial cells of forming capillaries: An autocrine mechanism contributing to angiogenesis, J. Cell. Biol. 141(7):1659-73 [1998]).
PTTG regulates bFGF mRNA and protein secretion, and this function requires a preserved C-terminus P-X-X-P motif. (Zhang, X. et al. [1999a]). It has also been reported that rat pituitary pttg is regulated in vivo and in vitro by estrogen, and the maximal induction of rat pituitary pttg mRNA in vivo occurred early in pituitary transformation (normal cell to hypertrophic/hyperplastic cell), coincident with bFGF and, vascular endothelial growth factor (VEGF) induction, and pituitary angiogenesis. (Heaney, A. P. et al. [1999]).
There remains a need for a therapeutic treatment for estrogen-sensitive neoplasms, such as breast and ovarian cancers, which can inhibit neoplastic cellular proliferation and/or transformation associated with PTTG overexpression. This and other benefits are provided by the present invention as described herein.